Methods and devices for the long-term culture of hematopoietic progenitor cells

ABSTRACT

The invention pertains to methods and devices for the long term, in vitroculture of hematopoietic progenitor cells in the absence of exogenously added hematopoietic growth factors, improved methods for the introduction of foreign genetic material into cells of hematopoietic origin, and to apparatus for performing these methods. The hematopoietic progenitor cells are cultured on a three-dimensional porous biomaterial. The three-dimensional porous biomaterial enhances hematopoietic progenitor cell survival and leads to an expansion of progenitor cell numbers and/or functionality, while maintaining progenitor cell pluripotency in the absence of exogenous growth factors. In addition, the three-dimensional porous biomaterial supports high level transduction on cells cultured upon such environment.

RELATED APPLICATIONS

This application is a divisional of pending application Ser. No.09/509,379, filed on Jun. 7, 2000, entitled METHODS AND DEVICES FOR THELONG-TERM CULTURE OF HEMATOPOIETIC PROGENITOR CELLS, which is theNational Stage filing of PCT/US98/20123 application filed on Sep. 25,1998, and which in turn claims priority from U.S. Provisionalapplication ser. No. 60/059,954 filed on Sep. 25, 1997. The contents ofthe foregoing applications are hereby expressly incorporated byreference.

GOVERNMENT SUPPORT

This work was funded in part under contract DAAH01-97-C-R121 from theU.S. Army Aviation and Missile Command. Accordingly, the United StatesGovernment may have certain rights to this invention.

FIELD OF THE INVENTION

This invention relates generally to hematopoietic cells, and morespecifically to methods and devices for long-term in vitro culturing ofhematopoietic progenitor cells, as well as methods for the introductionof exogenous genetic material into cells of hematopoietic origin.

BACKGROUND OF THE INVENTION

The circulating blood cells, such as erythrocytes, leukocytes, plateletsand lymphocytes, are the products of the terminal differentiation ofrecognizable precursors. In fetal life, hematopoiesis occurs throughoutthe reticular endothelial system. In the normal adult, terminaldifferentiation of the recognizable precursors occurs exclusively in themarrow cavities of the axial skeleton, with some extension into theproximal femora and humeri. These precursor cells, in turn, derive fromvery immature cells, called progenitors, which are assayed by theirdevelopment into contiguous colonies of mature blood cells in 1-3 weekcultures in semi-solid media, such as methylcellulose.

There have been reports of the isolation and purification ofhematopoietic progenitor cells (see, e.g., U.S. Pat. No. 5,061,620 asrepresentative), but such methods have not allowed for the long-termculture of such cells that maintain their viability and pluripotency.

Studies of the murine hematopoietic system in the murine bone marrowhave resulted in a detailed understanding of the murine system. Inaddition, retroviral gene transfer into cultured mouse bone marrow cellshas been made possible. While it has been possible to transferretroviral genes into cultured mouse bone marrow cells, the efficiencyof gene transfer into human bone marrow cells has been disappointing todate, which may reflect the fact that human long-term bone marrowcultures have been limited both in their longevity and more importantlyin their ability to maintain hematopoietic progenitor cell survival andpluripotentiality over time.

Human bone marrow cultures initially were found to have a limitedhematopoietic potential, producing decreasing numbers of hematopoieticprogenitor and mature blood cells, with cell production ceasing by sixto eight weeks. Subsequent modifications of the original system resultedonly in minor improvements. This has been largely attributed to thedependence of the hematopoietic progenitor cells upon environmentalinfluences such essential growth factors (hematopoietic growth factorsand cytokines) found in vivo. In addition to these factors, interactionswith cell surface molecules and extracellular matrix may be importantfor hematopoietic progenitor cell survival and proliferation. Previousefforts to advance in vitro proliferation and differentiation ofhematopoietic progenitor cells, examined the effects of cytokines invarious substrates, including pre-seeded stroma and fibronectin. Theaddition of exogenous growth factors to the culture environment,particularly IL-3 (Interleukin-3) and GM-CSF (GranulocyteMacrophage-Colony Stimulating, Factor), may lead to selective expansionof specific lineages. These findings suggest that addition of exogenousgrowth factors into hematopoietic progenitor cell cultures may adverselyaffect the multipotency of primitive hematopoietic progenitor cells bycausing them to differentiate and thus depleting the immaturehematopoietic progenitor population.

Alternative approaches have used irradiated bone marrow stroma to seedhematopoietic progenitor cells and have been shown to maintain thesecells in long-term culture initiating cells (LTCICs) and to increasetransduction of hematopoietic progenitor cells and LTCICs by retroviralvectors. However, questions have been raised about the risks ofinfection and immune reaction to transplantation of non-autologous bonemarrow. Fibronectin, a cellular stromal component, reduces this risk ofinfection and immune mediated response while enhancing retroviraltransduction. However, fibronectin alone may not be sufficient tomaintain primitive hematopoietic progenitor cells in vitro.

The hypothesis that the three-dimensional micro-environment of the bonemarrow plays a role in maintaining hematopoietic stem cell viability andpluripotency has led to investigating structures which mimic thistopography. Three-dimensional polymer devices (e.g., nylon mesh) havebeen shown to support hematopoietic progenitor cell survival,proliferation and multilineage differentiation, but require the presenceof growth factors. Such factors can be added exogenously, or suppliedvia secreting stromal cells which are co-cultured with the progenitorcells, or through the addition of stromal cell conditioned medium.

Hematopoietic progenitor cell expansion for bone marrow transplantationis a potential application of human long-term bone marrow cultures.Human autologous and allogeneic bone marrow transplantation arecurrently used as therapies for diseases such as leukemia, lymphoma, andother life-threatening diseases. For these procedures, however, a largeamount of donor bone marrow must be removed to ensure that there areenough cells for engraftment.

An approach providing hematopoietic progenitor cell expansion wouldreduce the need for large bone marrow donation and would make possibleobtaining a small marrow donation and then expanding the number ofprogenitor cells in vitro before infusion into the recipient. Also, itis known that a small number of hematopoietic progenitor cells circulatein the blood stream. If these cells could be selected and expanded, thenit would be possible to obtain the required number of hematopoieticprogenitor cells for transplantation from peripheral blood and eliminatethe need for bone marrow donation.

Hematopoietic progenitor cell expansion would also be useful as asupplemental treatment to chemotherapy and is another application forhuman long-term bone marrow cultures. Most chemotherapy agents act bykilling all cells going through cell division. Bone marrow is one of themost prolific tissues in the body and is therefore often the organ thatis initially damaged by chemotherapy drugs. The result is that bloodcell production is rapidly destroyed during chemotherapy treatment, andchemotherapy must be terminated to allow the hematopoietic system toreplenish the blood cell supplies before a patient is re-treated withchemotherapy.

A successful approach providing hematopoietic progenitor cell expansionwould greatly facilitate the production of a large number of furtherdifferentiated precursor cells of a specific lineage, and in turnprovide a larger number of differentiated hematopoietic cells with awide variety of applications, including blood transfusions.

Gene therapy is a rapidly growing field in medicine with an enormousclinical potential. Traditionally, gene therapy has been defined as aprocedure in which an exogenous gene is introduced into the cells of apatient in order to correct an inborn genetic error. Research in genetherapy has been ongoing for several years in several types of cells invitro and in animal studies, and more recently a number of clinicaltrials have been initiated.

The human hematopoietic system is an ideal choice for gene therapy inthat hematopoietic stem cells are readily accessible for treatment (bonemarrow or peripheral blood harvest) and they are believed to possessunlimited self-renewal capabilities (incurring lifetime therapy), andupon reinfusion, can expand and repopulate the marrow. Unfortunately,achieving therapeutic levels of gene transfer into stem cells has yet tobe accomplished in humans. The problem which remains to be addressed forsuccessful human gene therapy is the ability to insert the desiredtherapeutic gene into the chosen cells in a quantity such that it willbe beneficial to the patient. To date, methods for the efficientintroduction of exogenous genetic material into human hematopoietic stemcells have been limited.

There exists a need to influence favorably hematopoietic progenitor cellviability and pluripotency under long-term culture in vitro.

There exists a need to provide large numbers of differentiatedhematopoietic cells.

There also exists the need to improve the efficiency of exogenousgenetic material transfer into hematopoietic progenitor cells.

An object of the invention is to provide methods and devices that extendthe in vitro viability of hematopoietic stem cells while maintaining thehematopoietic progenitor cell properties of self-renewal andpluripotency.

Another object of the invention is to provide methods and devices forthe controlled production in large numbers of specific lineages ofprogenitor cells and their more differentiated hematopoietic cells.

Yet another object of the invention is to provide improved methods forgene transfer and transduction into cells of hematopoietic origin andhematopoietic progenitor cells in particular. These and other objects ofthe invention will be described in greater detail below.

SUMMARY OF THE INVENTION

The invention, in one important part, involves improved methods forculturing hematopoietic progenitor cells, which methods can, forexample, increase the period over which an amount of hematopoieticprogenitor cells can be cultured. Thus, one aspect of the invention isimproved preservation of a culture of hematopoietic progenitor cells.Another aspect is an improvement in the number of progeny that can beobtained from a sample of hematopoietic progenitor cells. Still anotheraspect of the invention is an improvement in the number ofdifferentiated progeny blood cells that can be obtained from a sample ofhematopoietic progenitor cells.

Surprisingly, according to the invention, it has been discovered thathematopoietic progenitor cells can be cultured without exogenous growthagents for extended periods of time, thereby increasing the supply ofhematopoietic progenitor cells and inhibiting the induction ofdifferentiation and/or the loss of progenitor cells during culture.Thus, the present invention permits the culture of hematopoieticprogenitor cells in vitro for more than 5 weeks, and even more than 6, 7or 8 weeks, without adding hematopoietic growth factors, inoculatedstromal cells or stromal cell conditioned medium. This is achieved,simply, by culturing the hematopoietic progenitor cells in a poroussolid scaffold.

According to one aspect of the invention, a method for in vitro cultureof hematopoietic progenitor cells is provided. An amount ofhematopoietic progenitor cells is introduced to a porous, solid matrixhaving interconnected pores of a pore size sufficient to permit thecells to grow throughout the matrix. The cells are cultured upon andwithin the matrix in an environment that is free of inoculated stromalcells, stromal cell conditioned medium, and exogenously addedhematopoietic growth factors that promote hematopoietic celldifferentiation, other than serum. The porous matrix can be one that isan open cell porous matrix having a percent open space of at least 50%,and preferably at least 75%. In one embodiment the porous solid matrixhas pores defined by interconnecting ligaments having a diameter atmidpoint, on average, of less than 150 μm. Preferably the porous solidmatrix is a metal-coated reticulated open cell foam of carbon containingmaterial, the metal coating being selected from the group consisting oftantalum, titanium, platinum (including other metals of the platinumgroup), niobium, hafnium, tungsten, and combinations thereof. Inpreferred embodiments, whether the porous solid matrix is metal-coatedor not, the matrix is coated with a biological agent selected from thegroup consisting of collagens, fibronectins, laminins, integrins,angiogenic factors, anti-inflammatory factors, glycosaminoglycans,vitrogen, antibodies and fragments thereof, functional equivalents ofthese factors, and combinations thereof. Most preferably the metalcoating is tantalum coated with a biological agent. In certain otherembodiments the porous solid matrix having seeded hematopoieticprogenitor cells and their progeny is impregnated with a gelatinousagent that occupies pores of the matrix.

The preferred embodiments of the invention are solid, unitarymacrostructures, i.e. not beads or packed beads. They also involvenonbiodegradable materials.

In other embodiments, before the introducing step, the hematopoieticprogenitor cells are obtained from a blood product. Preferably the bloodproduct is unfractionated bone marrow. In still other embodiments, themethod further includes the step of harvesting hematopoietic cells.Preferably, there is a first harvesting after a first culturing periodand at least one additional harvesting after at least one additionalculturing period. The harvested cells then are cultured in at least oneof an exogenously added agent selected from the group consisting of ahematopoietic growth factor that promotes hematopoietic cellmaintenance, expansion and/or differentiation, inoculated stromal cells,and stromal cell conditioned medium.

According to any of the foregoing embodiments, the method of theinvention can include, in said first culturing step, culturing the cellsin an environment that is free of hematopoietic progenitor cell survivaland proliferation factors such as interleukins 3, 6 and 11, stem cellligand and FLT-3 ligand. As mentioned above, the inventors havediscovered, surprisingly, that hematopoietic progenitor cells can begrown for extended periods of time without the addition of any of theseagents which typically are added in the prior art in order to preventthe hematopoietic progenitor cells from dying within several weeks.Still another embodiment of the invention is performing the firstculturing step in an environment that is free altogether of anyexogenously added hematopoietic progenitor cell growth factors, otherthan serum.

As will be understood, according to the invention, it is possible now toculture hematopoietic progenitor cells for 6, 7 or 8 weeks, and toharvest hematopoietic progenitor cells during this time interval forsubsequent exposure to culture conditions containing hematopoieticgrowth factors that promote hematopoietic cell maintenance, expansionand/or differentiation. Culturing and harvesting over this time periodis an independent aspect of the invention.

According to another aspect of the invention, a method is provided forin vitro culture of hematopoietic progenitor cells to producedifferentiated cells of hematopoietic origin. In a first culturing step,a first amount of hematopoietic progenitor cells is cultured in anenvironment that is free of inoculated stromal cells, stromal cellcondition medium and exogenously added hematopoietic growth factors thatpromote hematopoietic cell maintenance, expansion and/ordifferentiation, other than serum, under conditions and for a period oftime to increase the number of cultured hematopoietic progenitor cellsrelative to said first amount or to increase the functionality of thehematopoietic progenitor cells, thereby producing a second amount ofhematopoietic progenitor cells. Then, in a second culturing step, atleast a portion of the second amount of cultured hematopoieticprogenitor cells is cultured in an environment that includes at leastone of an agent selected from the group consisting of a hematopoieticgrowth factor that promotes hematopoietic cell maintenance, expansionand/or differentiation, inoculated stromal cells and stromal cellconditioned medium, to produce differentiated cells of hematopoieticorigin. In one embodiment, the environment is free of hematopoieticgrowth factors that promote survival and proliferation of hematopoieticprogenitor cells such as interleukins 3, 6 and 11, stem cell ligand andFLT-3 ligand. In another embodiment, the environment of the firstculturing step is free of any hematopoietic growth factors other thanthose present as a result of the addition of serum to the nutritivemedium. In this aspect of the invention, the method further can comprisea second culturing step which is a plurality of second culturing steps,each comprising culturing only a portion of the second amount ofhematopoietic progenitor cells. The method also can involve a harvestingstep between the first and second culturing steps, wherein theharvesting step comprises harvesting the at least a portion of thesecond amount prior to culturing the at least a portion of the secondamount in the second culturing step. The harvesting step also can be aplurality of harvesting steps spaced apart in time and, in thisinstance, the second culturing step can be a plurality of secondculturing steps, one for each of the harvesting steps. The preferredsource of the hematopoietic progenitor cells and the preferredconfiguration of the porous solid matrix is as described above.

According to another aspect of the invention, a method is provided forin vitro culture of hematopoietic progenitor cells to producedifferentiated cells of hematopoietic origin. In a first culturing step,hematopoietic progenitor cells are cultured in an environment that isfree of inoculated stromal cells, stromal cell condition medium andexogenously added hematopoietic growth factors that promotedifferentiation, other than serum, to generate cultured hematopoieticprogenitor cells. A portion of the cultured hematopoietic progenitorcells are harvested intermittently to generate a plurality ofintermittently harvested portions of cultured hematopoietic cells. Then,in a plurality of second culturing steps, the plurality ofintermittently cultured harvested portions are cultured in anenvironment that includes at least one agent selected from the groupconsisting of a hematopoietic growth factor that promotesdifferentiation, inoculated stromal cells and stromal cell conditionedmedium, to produce differentiated cells of hematopoietic origin. In oneembodiment, the environment of the first culturing step is free ofhematopoietic growth factors that promote survival and proliferation ofhematopoietic progenitor cells, such as interleukins 3, 6 and 11, stemcell ligand and FLT-3 ligand. In another embodiment, the environment ofthe first culturing step is free of any hematopoietic growth factors,other than those present as a result of the addition of serum to thenutritive medium. In this aspect of the invention, the preferred sourceof hematopoietic progenitor cells and the preferred porous solid matrixare as described above.

According to another aspect of the invention, a method is provided fortransducing exogenous genetic material into cells of hematopoieticorigin. Hematopoietic cells are cultured in a porous solid matrix havinginterconnected pores of a pore size sufficient to permit the cells togrow throughout the matrix. The cells are transduced with the exogenousgenetic material in situ on and within the matrix. It has been found,surprisingly, that the efficiency of transfer of genetic material whencarried out with the cells cultured upon the matrix is unexpectedlyincreased. The characteristics of various embodiments of the preferredporous solid matrices are as described above. Also, in this embodiment,the hematopoietic cells can be hematopoietic progenitor cells and thecells, whether progenitor or not, can be cultured in environments freeof factors that promote differentiation, factors that promote survivaland proliferation, any hematopoietic growth factors whatsoever,inoculated stromal cells or stromal cell conditioned media.

According to still another aspect of the invention, an apparatus forculturing cells is provided. The apparatus includes a first cell culturechamber containing a porous solid matrix having interconnected pores ofa pore size sufficient to permit cells to grow throughout the matrix.The apparatus also includes a second cell culture chamber. A conduitprovides fluid communication between the first and second cell culturechambers. A collection chamber is located between the first and secondcell culture chambers, the collection chamber interrupting fluidcommunication between the first and second cell culture chambers via theconduit. A first inlet valve on one side of the collection chamber isfor providing fluid to be received from the first cultured chamber intothe collection chamber. An outlet valve on the other side of thecollection chamber provides fluid to be received into the secondcultured chamber from the collection chamber. Finally, there is a secondinlet valve for the collection chamber for introducing a desired fluidinto the collection chamber, other than fluid from the first cellculture chamber, whereby fluid may be intermittently removed from thefirst cell culture chamber and provided to the second cell culturechamber without contamination of the first culture chamber by fluid fromthe second culture chamber.

According to yet another aspect of the invention, another apparatus forculturing cells is provided. This apparatus includes a first cellculture chamber containing a porous solid matrix having interconnectedpores of a pore size sufficient to permit cells to grow throughout thematrix. An inlet valve on the first cell culture chamber is provided forintroducing culture medium into the first cell culture chamber. A secondcell culture chamber also is provided, the first and second cell culturechambers being in fluid communication with one another via a conduit. Avalve on the conduit is provided for controlling fluid flow between thefirst and second cell culture chambers.

In either of the foregoing apparatus, the second cell culture chambercan be provided with a porous solid matrix having interconnected poresof a pore size sufficient to permit cells to grow throughout the matrix.Various embodiments are provided, wherein the porous solid matrix hasone or more of the preferred characteristics as described above. Inaddition, the various cell culture chambers can have ports and conduitsfor sampling material within the cell culture chamber, for augmentationby delivery of various agents to one or the other of the cell culturechambers and for controlling and permitting the continuous flow ofmedium through either or both of the cell culture chambers.

In yet another aspect of the invention, a solid porous matrix isprovided wherein hematopoietic progenitor cells, with or without theirprogeny, are attached to the solid porous matrix. In some embodiments,stromal cells may also be attached to the matrix. The porous matrix canbe one that is an open cell porous matrix having a percent open space ofat least 50%, and preferably at least 75%. In one embodiment the poroussolid matrix has pores defined by interconnecting ligaments having adiameter at midpoint, on average, of less than 150 μm. Preferably theporous solid matrix is a metal-coated reticulated open cell foam ofcarbon containing material, the metal coating being selected from thegroup consisting of tantalum, titanium, platinum (including other metalsof the platinum group), niobium, hafnium, tungsten, and combinationsthereof. In preferred embodiments, whether the porous solid matrix ismetal-coated or not, the matrix is coated with a biological agentselected from the group consisting of collagens, fibronectins, laminins,integrins, angiogenic factors, anti-inflammatory factors,glycosaminoglycans, vitrogen, antibodies and fragments thereof,functional equivalents of these factors, and combinations thereof. Mostpreferably the metal coating is tantalum coated with a biological agent.In certain other embodiments the porous solid matrix having seededhematopoietic progenitor cells and their progeny is impregnated with agelatinous agent that occupies pores of the matrix.

According to another aspect of the invention, a method for in vivomaintenance, expansion and/or differentiation of hematopoieticprogenitor cells is provided. The method involves implanting into asubject a porous, solid matrix having pre-seeded hematopoieticprogenitor cells and hematopoietic progenitor cell progeny. The porousmatrix has interconnected pores of a pore size sufficient to permit thecells to grow throughout the matrix and is an open cell porous matrixhaving a percent open space of at least 50%, and preferably at least75%. Various embodiments are provided, wherein the porous solid matrixhas one or more of the preferred characteristics as described above. Incertain other embodiments, the porous solid matrix further compriseshematopoietic progenitor cells and their progeny which are attached tothe matrix by introducing in vitro an amount of hematopoietic progenitorcells into the porous solid matrix, and culturing the hematopoieticprogenitor cells in an environment that is free of inoculated stromalcells, stromal cell conditioned medium, and exogenously addedhematopoietic growth factors that promote hematopoietic cellmaintenance, expansion and/or differentiation, other than serum. In yetother embodiments the porous solid matrix having seeded hematopoieticprogenitor cells and their progeny is impregnated with a gelatinousagent that occupies pores of the matrix.

In any of the foregoing embodiments involving hematopoietic cellmaintenance, expansion and/or differentiation using a hematopoieticgrowth factor, the hematopoietic growth factor used is selected from thegroup consisting of interleukin 3, interleukin 6, interleukin 7,interleukin 11, interleukin 12 stem cell factor, FLK-2 ligand, FLT-2ligand, Epo, Tpo, GMCSF, GCSF, Oncostatin M, and MCSF.

These and other aspects of the invention are described in greater detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a cell culture apparatusaccording to the invention.

FIG. 2. Survival and expansion of CD34⁺ HPCs in Cellfoam v. controlsystems at 1 week.

FIG. 3. Survival and expansion of CD34⁺ HPCs in Cellfoam v. controlsystems at 3 and 6 weeks.

FIG. 4. CFU ability of HSCs isolated from Cellfoam and control cultures.

FIG. 5. CD45⁺ cell number at 1, 3 and 6 weeks in Cellfoam and BMScultures supplemented with cytokines.

FIG. 6. CD45⁺ cell number at 1, 3 and 6 weeks in Cellfoam and BMSsupplemented with the combination cytokines.

FIG. 7. Fold difference of CD45⁺34⁺ cell yield in Cellfoam cultures ascompared to BMS and plastic control cultures at 3 and 6 weeks atnanogram (top) and picogram (bottom) concentrations.

FIG. 8. Total colony activity of cells isolated from Cellfoam andplastic cultures supplemented with cytokines.

FIG. 9. Fold difference of total colony activity in Cellfoam cultures ascompared to plastic control cultures at 3 and 6 weeks in nanogramconcentration supplementation experiments.

DETAILED DESCRIPTION OF THE INVENTION

The invention in one aspect involves culturing hematopoietic progenitorcells in a porous solid matrix without exogenous growth agents.

A porous, solid matrix, is defined as a three-dimensional structure with“sponge-like” continuous pores forming an interconnecting network. Thematrix can be rigid or elastic, and it provides a scaffold upon whichcells can grow throughout. Its pores are interconnected and provide thecontinuous network of channels extending through the matrix and alsopermit the flow of nutrients throughout. A preferred matrix is an opencell foam matrix having a percent open space of at least 50% andpreferably 75%. Thus, it is preferred that the open space comprise themajority of the matrix. This is believed to maximize cell migration,cell-cell contact, space for cell growth and accessibility to nutrients.It is preferred that the porous matrix be formed of a reticulated matrixof ligaments which at their center point are less than 150 μm indiameter, preferably 60 μm, whereby a cell can reside on or interactwith a portion of the ligament. Preferably, the average pore diameter ison the order of 300 μm, which resembles cancellous bone. Suitablematrices can be obtained using a number of different methods. Examplesof such methods include solvent casting or extraction of polymers, tracketching of a variety of materials, foaming of a polymer, thereplamineform process for hydroxyapatite, and other methodologies wellknown to those of ordinary skill in the art. The materials employed canbe natural or synthetic, including biological materials such asproteins, hyaluronic acids, synthetic polymers such as polyvinylpyrolidones, polymethylmethacrylate, methyl cellulose, polystyrene,polypropylene, polyurethane, ceramics such as tricalcium phosphate,calcium aluminate, calcium hydroxyapatite and ceramic-reinforced orcoated polymers. If the starting material for the scaffold is not metal,a metal coating can be applied to the three-dimensional matrix. Metalcoatings provide further structural support and/or cell growth andadhesive properties to the matrix. Preferred metals used as coatingscomprise tantalum, titanium, platinum and metals in the same elementgroup as platinum, niobium, hafnium, tungsten, and combinations ofalloys thereof. Coating methods for metals include a process such as CVD(Chemical Vapor Deposition). The preferred matrix, refered to hereinthroughout as Cellfoam, is described in detail in U.S. Pat. No.5,282,861, and is incorporated herein by reference. More specifically,the preferred matrix is a reticulated open cell substrate formed by alightweight, substantially rigid foam of carbon-containing materialhaving open spaces defined by an interconnecting network, wherein saidfoam material has interconnected continuous channels, and a thin film ofmetallic material deposited onto the reticulated open cell substrate andcovering substantially all of the interconnecting network to form acomposite porous biocompatible material creating a porous microstructuresimilar to that of natural cancellous bone.

Additionally, such matrices can be coated with biological agents whichcan promote cell adhesion for the cultured hematopoietic cells, allowingfor improved migration, growth and proliferation. Moreover, when thesematrices are used for the in vivo maintenance, expansion and/ordifferentiation of hematopoietic progenitor cells (i.e., when thematrices with the cells are implanted into a subject, —see alsodiscussion below), biological agents that promote angiogenesis(vascularization) and biological agents that prevent/reduce inflammationmay also be used for coating of the matrices. Preferred biologicalagents comprise collagens, fibronectins, laminins, integrins, angiogenicfactors, anti-inflammatory factors, glycosaminoglycans, vitrogen,antibodies and fragments thereof, functional equivalents of theseagents, and combinations thereof.

Angiogenic factors include platelet derived growth factor (PDGF),vascular endothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), bFGF-2, leptins, plasminogen activators (tPA, uPA),angiopoietins, lipoprotein A, transforming growth factor-β, bradykinin,angiogenic oligosaccharides (e.g., hyaluronan, heparan sulphate),thrombospondin, hepatocyte growth factor (also known as scatter factor)and members of the CXC chemokine receptor family. Anti-inflammatoryfactors comprise steroidal and non-steroidal compounds and examplesinclude: Alclofenac; Alclometasone Dipropionate; Algestone Acetonide;Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol; Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

In certain embodiments of the invention the porous solid matrix havingseeded hematopoietic progenitor cells, with or without their progeny, isimpregnated with a gelatinous agent that occupies pores of the matrix.By “seeded” it is meant that the hematopoietic progenitor cells, with orwithout their progeny, are seeded prior to, substantially at the sametime as, or following impregnation (or infiltration) with a gelatinousagent. For example, the cells may be mixed with the gelatinous agent andseeded at the same time as the the impregnation of the matrix with theagent. In some embodiments, the hematopoietic progenitor cells, with orwithout their progeny, are pre-seeded onto the porous solid matrix.According to the invention, an amount of the cells is introduced invitro into the porous solid matrix, and cultured in an environment thatis free of inoculated stromal cells, stromal cell conditioned medium,and exogenously added hematopoietic growth factors that promotehematopoietic cell maintenance, expansion and/or differentiation, otherthan serum.

“Impregnation” with a gelatinous agent serves as to contain the cellswithin the matrix, and also to help maintain and/or enhance cellattachment onto the matrix. The “gelatinous” agent may be one that canbe maintained in a fluid state initially, and after its application intothe matrix, be gelatinized in situ in the matrix. Such gelatinizationmay to occur in a number of different ways, including altering theagent's temperature, irradiating the agent with an energy source (e.g.,light), etc. The agent may exist in a continuum from a fluid state to asemi-solid (gelatinous) state to a solid state. An agent's final stateand gelatinization will always depend upon the particular “gelatinous”agent used and its individual properties. A preferred “gelatinous” agentis characterized also by its structural porosity, necessary for allowingthe nutrients of the growth media to reach the cells throughout thematrix. Exemplary “gelatinous” agents include cellulosic polysaccharides(such as cellulose, hemicellulose, methylcellulose, and the like), agar,agarose, albumin, algal mucin, mucin, mucilage, collagens,glycosaminoglycans, and proteoglycans (including their sulphated forms).In certain embodiments, the gelatinous agent may impregnate the matrixcompletely, in some embodiments partially, and in other embodimentsminimally, serving only as a coating of the outer surfaces of thematrix. The extent of the impregnation will largely depend upon thephysical characteristics of the “gelatinous” agent of choice. Inpreferred embodiments the “gelatinous” agent is methylcellulose and theimpregnation is complete.

The cells cultured according to the methods of the invention arehematopoietic progenitor cells. “Hematopoietic progenitor cells” as usedherein refers to immature blood cells having the capacity to self-renewand to differentiate into the more mature blood cells (also describedherein as “progeny”) comprising granulocytes (e.g., promyelocytes,neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes,erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producingmegakaryocytes, platelets), and monocytes (e.g., monocytes,macrophages). It is known in the art that such cells may or may notinclude CD34⁺ cells. CD34⁺ cells are immature cells present in the“blood products” described below, express the CD34 cell surface marker,and are believed to include a subpopulation of cells with the“progenitor cell” properties defined above.

The hematopoietic progenitor cells can be obtained from blood products.A “blood product” as used in the present invention defines a productobtained from the body or an organ of the body containing cells ofhematopoietic origin. Such sources include unfractionated bone marrow,umbilical cord, peripheral blood, liver, thymus, lymph and spleen. Itwill be apparent to those of ordinary skill in the art that all of theaforementioned crude or unfractionated blood products can be enrichedfor cells having “hematopoietic progenitor cell” characteristics in anumber of ways. For example, the blood product can be depleted from themore differentiated progeny. The more mature, differentiated cells canbe selected against, via cell surface molecules they express.Additionally, the blood product can be fractionated selecting for CD34⁺cells. As mentioned earlier, CD34⁺ cells are thought in the art toinclude a subpopulation of cells capable of self-renewal andpluripotentiality. Such selection can be accomplished using, forexample, commercially available magnetic anti-CD34 beads (Dynal, LakeSuccess, N.Y.). Unfractionated blood products can be obtained directlyfrom a donor or retrieved from cryopreservative storage.

Employing the culture conditions described in greater detail below, itis possible according to the invention to preserve hematopoieticprogenitor cells and to stimulate the expansion of hematopoieticprogenitor cell number and/or colony forming unit potential. Onceexpanded, the cells, for example, can be returned to the body tosupplement, replenish, etc. a patient's hematopoietic progenitor cellpopulation. This might be appropriate, for example, after an individualhas undergone chemotherapy. There are certain genetic conditions whereinhematopoietic progenitor cell numbers are decreased, and the methods ofthe invention may be used in these situations as well.

It also is possible to take the increased numbers of hematopoieticprogenitor cells produced according to the invention and stimulate themwith hematopoietic growth agents that promote hematopoietic cellmaintenance, expansion and/or differentiation, to yield the more matureblood cells, in vitro. Such expanded populations of blood cells may beapplied in vivo as described above, or may be used experimentally aswill be recognized by those of ordinary skill in the art. Suchdifferentiated cells include those described above, as well as T cells,plasma cells, erythrocytes, megakaryocytes, basophils, polymorphonuclearleukocytes, monocytes, macrophages, eosinohils and platelets.

In the preferred embodiments of the invention, the hematopoieticprogenitor cells are continuously cultured for an extended period oftime, and aliquots of the cultured cells are harvested spaced apart intime or intermittently. “Harvesting hematopoietic cells” is defined asthe dislodging or separation of cells from the matrix. This can beaccomplished using a number of methods, such as enzymatic, centrifugal,electrical or by size, or the one preferred in the present invention, byflushing of the cells using the media in which the cells are incubated.The cells can be further collected and separated. “Harvesting stepsspaced apart in time” or “intermittent harvest of cells” is meant toindicate that a portion of the cells are harvested, leaving behindanother portion of cells for their continuous culture in the establishedmedia, maintaining a continuous source of the original cells and theircharacteristics. Harvesting “at least a portion of” means harvesting asubpopulation of or the entirety of. Thus, as will be understood by oneof ordinary skill in the art, the invention can be used to expand thenumber of hematopoietic progenitor cells, all the while harvestingportions of those cells being expanded for treatment to develop evenlarger populations of differentiated cells.

In all of the culturing methods according to the invention, except asotherwise provided, the media used is that which is conventional forculturing cells. Examples include RPMI, DMEM, ISCOVES, etc. Typicallythese media are supplemented with human or animal plasma or serum. Suchplasma or serum can contain small amounts of hematopoietic growthfactors. The media used according to the present invention, however, candepart from that used conventionally in the prior art. In particular, ithas been discovered, surprisingly, that hematopoietic progenitor cellscan be cultured on the matrices described above for extended periods oftime without the need for adding any exogenous growth agents (other thanthose which may be contained in plasma or serum, hereinafter “serum”),without inoculating the environment of the culture with stromal cellsand without using stromal cell conditioned media. Prior to the presentinvention, at least one of the foregoing agents was believed necessaryin order to culture hematopoietic progenitor cells.

The growth agents of particular interest in connection with the presentinvention are hematopoietic growth factors. By hematopoietic growthfactors, it is meant factors that influence the survival, proliferationor differentiation of hematopoietic cells. Growth agents that affectonly survival and proliferation, but are not believed to promotedifferentiation, include the interleukins 3, 6 and 11, stem cell ligandand FLT-3 ligand. Hematopoietic growth factors that promotedifferentiation include the colony stimulating factors such as GMCSF,GCSF, MCSF, Tpo, Epo, Oncostatin M, and interleukins other than IL-3, 6and 11. The foregoing factors are well known to those of ordinary skillin the art. Most are commercially available. They can be obtained bypurification, by recombinant methodologies or can be derived orsynthesized synthetically.

In one aspect of the invention, the hematopoietic progenitor cells arecultured in an environment that is free of inoculated stromal cells,stromal cell conditioned medium and exogenously added hematopoieticgrowth factors that promote differentiation of hematopoietic cells,other than serum. By “inoculated” stromal cells, it is meant that thecell culture chamber is free of stromal cells which have been introducedinto the chamber as an inoculum for promoting survival, proliferation ordifferentiation of the hematopoietic progenitor cells, excluding,however, stromal cells which are contained naturally in the isolateblood product.

“Stromal cells” as used herein comprise fibroblasts and mesenchymalcells, with or without other cells and elements, and can be seeded priorto, or substantially at the same time as, the hematopoietic progenitorcells, therefore establishing conditions that favor the subsequentattachment and growth of hematopoietic progenitor cells. Fibroblasts canbe obtained via a biopsy from any tissue or organ, and include fetalfibroblasts. These fibroblasts and mesenchymal cells may be transfectedwith exogenous DNA that encodes, for example, one of the hematopoieticgrowth factors described above.

“Stromal cell conditioned medium” refers to medium in which theaforementioned stromal cells have been incubated. The incubation isperformed for a period sufficient to allow the stromal cells to secretefactors into the medium. Such “stromal cell conditioned medium” can thenbe used to supplement the culture of hematopoietic progenitor cellspromoting their proliferation and/or differentiation.

Thus, when cells are cultured without any of the foregoing agents, it ismeant herein that the cells are cultured without the addition of suchagent except as may be present in serum, ordinary nutritive media orwithin the blood product isolate, unfractionated or fractionated, whichcontains the hematopoietic progenitor cells.

The culture of the hematopoietic cells preferably occurs underconditions to increase the number of such cells and/or the colonyforming potential of such cells. The conditions used refer to acombination of conditions known in the art (e.g., temperature, CO₂ andO₂ content, nutritive media, etc.). The time sufficient to increase thenumber of cells is a time that can be easily determined by a personskilled in the art, and can vary depending upon the original number ofcells seeded. As an example, discoloration of the media can be used asan indicator of confluency. Additionally, and more precisely, differentvolumes of the blood product can be cultured under identical conditions,and cells can be harvested and counted over regular time intervals, thusgenerating the “control curves”. These “control curves” can be used toestimate cell numbers in subsequent occasions.

The conditions for determining colony forming potential are similarlydetermined. Colony forming potential is the ability of a cell to formprogeny. Assays for this are well known to those of ordinary skill inthe art and include seeding cells into a semi-solid, treating them withgrowth factors and counting the number of colonies.

According to another aspect of the invention a method for in vivomaintenance, expansion and/or differentiation of hematopoieticprogenitor cells is provided. The method involves implanting into asubject a porous solid matrix having pre-seeded hematopoietic progenitorcells and hematopoietic progenitor cell progeny. Implantation ofmatrices similar to the matrices of the invention is well known in theart (Stackpool, GJ, et al, Combined Orthopaedic Research SocietiesMeeting, Nov. 6-8, 1995, San Diego, Calif., Abstract Book p. 45; Turner,TM, et al., 21st Annual Meeting of the Society for Biomaterials, March18-22, San Francisco, Calif., Abstract Book p. 125). Such matrices arebiocompatible (i.e., no immune reactivity-no rejection) and can beimplanted and transplanted in a number of different tissues of asubject. Such methods are useful in a variety of ways, including thestudy of hematopoietic progenitor cell maintenance, expansion and/ordifferentiation in vivo, in a number of different tissues of a subject,or in different subjects.

As used herein, a subject is a human, non-human primate, cow, horse,pig, sheep, goat, dog, cat or rodent. Human hematopoietic progenitorcells and human subjects are particularly important embodiments. Asdescribed above, when the matrices of the invention are used for such invivo implantation studies, biological agents that promote angiogenesis(vascularization) and/or prevent/reduce inflammation may also be usedfor coating of the matrices. Preferred biological agents are asdescribed above. Also as described above, the hematopoietic progenitorcells are pre-seeded onto the porous solid matrix and cultured in vitroaccording to the invention, before implantation into a subject.According to the invention, an amount of the cells is introduced invitro into the porous solid matrix, and cultured in an environment thatis free of inoculated stromal cells, stromal cell conditioned medium,and exogenously added hematopoietic growth factors that promotehematopoietic cell maintenance, expansion and/or differentiation, otherthan serum. Implantation is then carried out.

The invention also involves the unexpected discovery that hematopoieticprogenitor cells can be more efficiently transduced if the transductionoccurs while the hematopoietic progenitor cells are on and within asolid porous matrix as described above. As used herein, “transduction ofhematopoietic cells” refers to the process of transferring exogenousgenetic material into a cell of hematopoietic origin. The terms“transduction”, “transfection” and “transformation” are usedinterchangeably throughout this letter, and refer to the process oftransferring exogenous genetic material into a cell. As used herein,“exogenous genetic material” refers to nucleic acids oroligonucleotides, either natural or synthetic, that are introduced intothe hematopoietic progenitor cells. The exogenous genetic material maybe a copy of that which is naturally present in the cells, or it may notbe naturally found in the cells. It typically is at least a portion of anaturally occuring gene which has been placed under operable control ofa promoter in a vector construct.

Various techniques may be employed for introducing nucleic acids intocells. Such techniques include transfection of nucleic acid-CaPO₄precipitates, transfection of nucleic acids associated with DEAE,transfection with a retrovirus including the nucleic acid of interest,liposome mediated transfection, and the like. For certain uses, it ispreferred to target the nucleic acid to particular cells. In suchinstances, a vehicle used for delivering a nucleic acid according to theinvention into a cell (e.g., a retrovirus, or other virus; a liposome)can have a targeting molecule attached thereto. For example, a moleculesuch as an antibody specific for a surface membrane protein on thetarget cell or a ligand for a receptor on the target cell can be boundto or incorporated within the nucleic acid delivery vehicle. Forexample, where liposomes are employed to deliver the nucleic acids ofthe invention, proteins which bind to a surface membrane proteinassociated with endocytosis may be incorporated into the liposomeformulation for targeting and/or to facilitate uptake. Such proteinsinclude proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half life, and the like. Polymeric delivery systems alsohave been used successfully to deliver nucleic acids into cells, as isknown by those skilled in the art. Such systems even permit oraldelivery of nucleic acids.

In the present invention, the preferred method of introducing exogenousgenetic material into hematopoietic cells is by transducing the cells insitu on the matrix using replication deficient retroviruses.Replication-deficient retroviruses are capable of directing synthesis ofall virion proteins, but are incapable of making infectious particles.Accordingly, these genetically altered retroviral vectors have generalutility for high-efficiency transduction of genes in cultured cells, andspecific utility for use in the method of the present invention.Retroviruses have been used extensively for transferring geneticmaterial into cells. Standard protocols for producingreplication-deficient retroviruses (including the steps of incorporationof exogenous genetic material into a plasmid, transfection of apackaging cell line with plasmid, production of recombinant retrovirusesby the packaging cell line, collection of viral particles from tissueculture media, and infection of the target cells with the viralparticles) are provided in the art.

The major advantage of using retroviruses is that the viruses insertefficiently a single copy of the gene encoding the therapeutic agentinto the host cell genome, thereby permitting the exogenous geneticmaterial to be passed on to the progeny of the cell when it divides. Inaddition, gene promoter sequences in the LTR region have been reportedto enhance expression of an inserted coding sequence in a variety ofcell types. The major disadvantages of using a retrovirus expressionvector are (1) insertional mutagenesis, i.e., the insertion of thetherapeutic gene into an undesirable position in the target cell genomewhich, for example, leads to unregulated cell growth and (2) the needfor target cell proliferation in order for the therapeutic gene carriedby the vector to be integrated into the target genome. Despite theseapparent limitations, delivery of a therapeutically effective amount ofa therapeutic agent via a retrovirus can be efficacious if theefficiency of transduction is high and/or the number of target cellsavailable for transduction is high.

Yet another viral candidate useful as an expression vector fortransformation of hematopoietic cells is the adenovirus, adouble-stranded DNA virus. Like the retrovirus, the adenovirus genome isadaptable for use as an expression vector for gene transduction, i.e.,by removing the genetic information that controls production of thevirus itself. Because the adenovirus functions usually in anextrachromosomal fashion, the recombinant adenovirus does not have thetheoretical problem of insertional mutagenesis. On the other hand,adenoviral transformation of a target hematopoietic cell may not resultin stable transduction. However, more recently it has been reported thatcertain adenoviral sequences confer intrachromosomal integrationspecificity to carrier sequences, and thus result in a stabletransduction of the exogenous genetic material.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable vectors are available for transferring exogenous geneticmaterial into hematopoietic cells. The selection of an appropriatevector to deliver a therapeutic agent for a particular conditionamenable to gene replacement therapy and the optimization of theconditions for insertion of the selected expression vector into thecell, are within the scope of one of ordinary skill in the art withoutthe need for undue experimentation. The promoter characteristically hasa specific nucleotide sequence necessary to initiate transcription.Optionally, the exogenous genetic material further includes additionalsequences (i.e., enhancers) required to obtain the desired genetranscription activity. For the purpose of this discussion an “enhancer”is simply any nontranslated DNA sequence which works contiguous with thecoding sequence (in cis) to change the basal transcription leveldictated by the promoter. Preferably, the exogenous genetic material isintroduced into the hematopoietic cell genome immediately downstreamfrom the promoter so that the promoter and coding sequence areoperatively linked so as to permit transcription of the coding sequence.A preferred retroviral expression vector includes an exogenous promoterelement to control transcription of the inserted exogenous gene. Suchexogenous promoters include both constitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a gene under the control of aconstitutive promoter is expressed under all conditions of cell growth.Exemplary constitutive promoters include the promoters for the followinggenes which encode certain constitutive or “housekeeping” functions:hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase(DHFR) (Scharfinann et al., Proc. Natl. Acad. Sci. USA 88:4626-4630(1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvatekinase, phosphoglycerol mutase, the actin promoter (Lai et al., Proc.Natl. Acad. Sci. USA 86: 10006-10010 (1989)), and other constitutivepromoters known to those of skill in the art. In addition, many viralpromoters function constitutively in eucaryotic cells. These include:the early and late promoters of SV40; the long terminal repeats (LTRS)of Moloney Leukemia Virus and other retroviruses; and the thymidinekinase promoter of Herpes Simplex Virus, among many others. Accordingly,any of the above-referenced constitutive promoters can be used tocontrol transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressedonly or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). Induciblepromoters include responsive elements (REs) which stimulatetranscription when their inducing factors are bound. For example, thereare REs for serum factors, steroid hormones, retinoic acid and cyclicAMP. Promoters containing a particular RE can be chosen in order toobtain an inducible response and in some cases, the RE itself may beattached to a different promoter, thereby conferring inducibility to therecombinant gene. Thus, by selecting the appropriate promoter(constitutive versus inducible; strong versus weak), it is possible tocontrol both the existence and level of expression of a therapeuticagent in the genetically modified hematopoietic cell. Selection andoptimization of these factors for delivery of a therapeuticallyeffective dose of a particular therapeutic agent is deemed to be withinthe scope of one of ordinary skill in the art without undueexperimentation, taking into account the above-disclosed factors and theclinical profile of the patient.

In addition to at least one promoter and at least one hetefologousnucleic acid encoding the therapeutic agent, the expression vectorpreferably includes a selection gene, for example, a neomycin resistancegene, for facilitating selection of hematopoietic cells that have beentransfected or transduced with the expression vector. Alternatively, thehematopoietic cells are transfected with two or more expression vectors,at least one vector containing the gene(s) encoding the therapeuticagent(s), the other vector containing a selection gene. The selection ofa suitable promoter, enhancer, selection gene and/or signal sequence(described below) is deemed to be within the scope of one of ordinaryskill in the art without undue experimentation.

The selection and optimization of a particular expression vector forexpressing a specific gene product in an isolated hematopoietic cell isaccomplished by obtaining the gene, preferably with one or moreappropriate control regions (e.g., promoter, insertion sequence);preparing a vector construct comprising the vector into which isinserted the gene; transfecting or transducing cultured hematopoieticcells in vitro with the vector construct; and determining whether thegene product is present in the cultured cells. TABLE 1 Human GeneTherapy Protocols Approved by RAC: 1990-1994 Severe combined Autologouslymphocytes transduced with human Jul. 31, 1990 immune deficiency ADAgene (SCID) due to ADA deficiency Advanced cancer Tumor-infiltratinglymphocytes transduced with tumor Jul. 31, 1990 necrosis factor geneAdvanced cancer Immunization with autologous cancer cells transducedOct. 07, 1991 with tumor necrosis factor gene Advanced cancerImmunization with autologous cancer cells transduced Oct. 07, 1991 withinterleukin-2 gene Asymptomatic patients Murine Retro viral vectorencoding HIV-1 genes Jun. 07, 1993 infected with HIV-1 [HIV-IT(V)] AIDSEffects of a transdominant form of rev gene on AIDS Jun. 07, 1993intervention Advanced cancer Human multiple-drug resistance (MDR) genetransfer Jun. 08, 1993 HIV infection Autologous lymphocytes transducedwith catalytic Sep. 10, 1993 ribozyme that cleaves HIV-1 RNA (Phase Istudy) Metastatic melanoma Genetically engineered autologous tumorvaccines Sep. 10, 1993 producing interleukin-2 HIV infection MurineRetro viral vector encoding HIV-IT(V) genes Dec. 03, 1993 (open labelPhase I/II trial) HIV infection Adoptive transfer of syngeneic cytotoxicT lymphocytes Mar. 03, 1994 (identical twins) (Phase I/II pilot study)Breast cancer (chemo- Use of modified Retro virus to introducechemotherapy Jun. 09, 1994 protection during resistance sequences intonormal hematopoietic cells therapy) (pilot study) Fanconi's anemia Retroviral mediated gene transfer of the Fanconi anemia Jun. 09, 1994complementation group C gene to hematopoietic progenitors Metastaticprostate Autologous human granulocyte macrophage-colony ORDA/NIHcarcinoma stimulating factor gene transduced prostate cancer vaccineAug. 03, 1994* *(first protocol to be approved under the acceleratedreview process; ORDA = Office of Recombinate DNA Activities) Metastaticbreast cancer In vivo infection with breast-targeted Retro viral vectorSep. 12, 1994 expressing antisense c-fox or antisense c-myc RNAMetastatic breast cancer Non-viral system (liposome-based) fordelivering human Sep. 12, 1994 (refractory or recurrent) interleukin-2gene into autologous tumor cells (pilot study) Mild Hunter syndromeRetro viral-mediated transfer of the iduronate-2-sulfatase Sep. 13, 1994gene into lymphocytes Advanced mesothelioma Use of recombinantadenovirus (Phase I study) Sep. 13, 1994

The foregoing (Table 1), represent only examples of genes that can bedelivered according to the methods of the invention. Suitable promoters,enhancers, vectors, etc., for such genes are published in the literatureassociated with the foregoing trials. In general, useful genes replaceor supplement function, including genes encoding missing enzymes such asadenosine deaminase (ADA) which has been used in clinical trials totreat ADA deficiency and cofactors such as insulin and coagulationfactor VIII. Genes which affect regulation can also be administered,alone or in combination with a gene supplementing or replacing aspecific function. For example, a gene encoding a protein whichsuppresses expression of a particular protein-encoding gene can beadministered. The invention is particularly useful in delivering geneswhich stimulate the immune response, including genes encoding viralantigens, tumor antigens, cytokines (e.g. tumor necrosis factor) andinducers of cytokines (e.g. endotoxin).

The invention also provides various apparatus for carrying out themethods of the invention. The preferred apparatus is depicted in FIG. 1.The principle components of the embodiment depicted in FIG. 1 are a pairof cell culture chambers, one for continuously culturing hematopoieticprogenitor cells in an environment which promotes the survival andproliferation of the progenitor cells, but not the differentiation ofthe progenitor cells. The other cell culture chamber (which can be oneor more second cell culture chambers) is for receiving intermittentlyportions of the cells cultured in the first cell culture chamber forculturing in an environment that includes growth factors that promotedifferentiation of hematopoietic progenitor cells.

Referring to FIG. 1, a first cell culture chamber 10, and a second cellculture chamber 12 are shown. The cell culture chambers 10, 12 havewalls defining the inside of the chamber. A connection conduit 16provides fluid communication between the first and second cell culturechambers. The connection conduit can be any fluid conduit between thefirst and second cell culture chambers, although in the embodimentdepicted, the connection conduit 16 includes a plurality of valves and acollection chamber, described in greater detail below. Each of the firstand second cell culture chambers 10, 12 contain a porous solid matrix18, as described in detail above. The porous solid matrix 18 issupported by matrix supports 20 which hold the matrix 18 away from thewalls 14 to provide a space 22 permitting circulation of mediathroughout the matrix 18. Preferably there is a seal which restrictsfluid flow around the matrix, forcing the fluid to flow through thematrix.

The first cell culture chamber 10 is provided with an inlet port 24which communicates with a media input conduit 26 for supplying media tothe first culture chamber. The port 24 or the media input conduit 26 canbe provided with a valve (not shown) for controlling the flow of mediainto the first cell culture chamber 10.

The first cell culture chamber has a top 28 which closes the first cellculture chamber. This top 28 may engage the walls 14 of the first cellculture chamber in a sealing fashion or, alternatively, can engage thewalls 14 of the first cell culture chamber in a manner to permit theexchange of gases as is conventional in certain cell culture apparatus.In the embodiment depicted, the top sealingly engages the walls. Asample port 30 is provided in the top 28 and communicates with asampling conduit 32 for permitting materials to be added into or removedfrom the first cell culture chamber. Preferably, as shown in connectionwith the second cell culture chamber and described in more detail below,a second conduit can be provided (an augmentation conduit), whereby thesample conduit is for removing material from the cell culture chamberwhereas the augmentation conduit is for introducing material into thecell culture chamber. The sample port 30 and/or sample conduit 32 can beprovided with a valve (not shown) for isolating the internal environmentof the first cell culture chamber from external environmentalinfluences.

The first cell culture chamber 10 also has an outlet port 34communicating with the connection conduit 16.

Turning to the second cell culture chamber 12, wherein like numeralsindicate like parts, the second cell culture chamber has walls 14 forcontaining a porous solid matrix 18 supported by matrix supports 20. Thetop 28 of the second cell culture chamber 12 is sealingly engaged withthe walls 14 of the second cell culture chamber. The top includes asample port 30 and a sample conduit 32 communicating with the sampleport 30 for obtaining samples of material from inside of the second cellculture chamber. The top 28 of the second cell culture chamber 12 alsoincludes an outlet port 34 communicating with an outlet conduit 36whereby, preferably, media may be circulated continuously throughout thesystem being introduced via the media input conduit 26 and leaving thesystem via the media outlet conduit 36. The second cell culture chamber12 also includes an augmentation conduit 38 for supplying the secondcell culture chamber with materials, preferably hematopoietic growthfactors that induce differentiation, to the second cell culture chamber.

Turning to the connection conduit 16, as mentioned above this can be anyconduit, and preferably there is at least one valve between the firstcell culture chamber 10 and the second cell culture chamber 12 alongthis conduit, whereby the flow of media between the first and secondcell culture chambers can be interrupted. In the embodiment depicted,the connection conduit 16 includes a first portion 40 exiting the firstcell-culture chamber 10 and terminating in fluid communication with acollection chamber 42. A second portion 44 of the connection conduitprovides fluid communication from the collection chamber 42 to thesecond cell culture chamber 12. The first portion 40 is interrupted by afirst portion valve 46 and the second portion 44 of the connectionconduit 16 is interrupted by a second portion valve 48.

The collection chamber also is in fluid communication with a flushingconduit 50 which has a flushing conduit valve 52.

In one embodiment of operating the apparatus of the invention, the valve(not shown) at inlet port 24 and outlet port 34, first portion valve 46,second portion valve 48, and outlet conduit valve (not shown) at theoutlet port 34 of second cell culture chamber are open. Valves (notshown) of the sample ports 30 of the first and second cell culturechambers 10, 12, valve (not shown) at the port of the second cellculture chamber 12 communicating with the augmentation conduit 38 isclosed and the flushing conduit valve 52 is closed. In this manner,media can be perfused through the first cell culture chamber, throughthe connection conduit and through the second cell culture chambercontinuously, if desired. As will be readily understood, mediaintroduced into the first cell culture chamber can be prevented fromcontacting the second cell culture chamber by closing valve 48 andopening valve 52. Likewise, the second cell culture chamber can receivemedia different from that received by the first cell culture chamber byopening the valve at the port communicating with the second cell culturechamber via the augmentation conduit 38, which may provide the onlymedia to the second cell culture chamber, may augment media receivedinto the second cell culture chamber from the first cell culture chamberor may augment media received via flushing conduit 50. In addition toproviding for the differential media requirement as describe above forthe first culturing step and the second culturing steps of theinvention, the present apparatus also provides for the transfer of cellsbetween the chambers via the conduit arrangement shown. In thisembodiment, a gentle pulse of fluid is applied to the first cell culturechamber, sufficient to dislodge hematopoietic progenitor cells from theporous solid matrix in the first culture chamber. These cells then canbe carried by fluid movement from the first cell culture chamber intothe collection chamber. The collection chamber can, if desired, beprovided with a means for temporarily maintaining the cells in thechamber such as by a moveable membrane, filter or the like, althoughsuch structure is not necessary for the operation of the apparatus ofthe invention. Once the cells are within the collection chamber 42,valve 46 can be closed. Subsequently, valve 52 can be opened, and thecells can be flushed by fluid pressure from the collection chamber 42into the second cell culture chamber 12. In this manner, as a result ofclosing valve 46, it is ensured that hematopoietic growth factors thathave been introduced into the second cell culture chamber do not flowbackwards into the first cell culture chamber, contaminating the firstcell culture chamber with unwanted material. The mere pressure due tocontinuous flow of media, however, may be sufficient to prevent backflowand the closing of valve 46 may be unnecessary. Numerous modificationsto the apparatus shown will be apparent to those of ordinary skill inthe art. The important aspects of the apparatus are the provision of twocell culture chambers and the mechanism for fluid communication betweenthem, with a valve arrangement, etc. whereby the first cell culturechamber cannot be contaminated with unwanted materials which are addeddownstream into the second cell culture chamber.

EXAMPLES

Experimental Procedures

Long-Term Cultures:

CD34⁺ hematopoietic progenitor cells were derived from human bone marrow(Poietic Technologies) isolated using magnetic anti-human CD34⁺ beads(Dynal, Lake Success, N.Y.) and separated from these beads using ananti-idiotype antibody (Detachabead, Dynal). All culture conditions wereseeded with 2×10⁵ cells to ensure enough cells for all analyses,particularly from control cultures. While preliminary data indicatedthat CD34⁺ cells would survive well in Cellfoam, it was anticipated thatculture in the absence of cytokines would lead to reduced cell numbersin control cultures. For the purposes of planning the experiments, weestimated that up to 75% of the cells (or 1.5×10⁵ cells) may be lost,leaving 5×10⁴ cells per reactor, enough cells to perform flow cytometry,multipotency colony assays and LTCIC (Long Term Culture Initiating Cell)analyses. Cultures were performed in duplicate to provide side by sidecomparisons of each culture time point. Thus, each culture time pointused two reactors, each seeded with 2×10⁵ CD34⁺ cells.

2×10⁵ CD34⁺ cells in 1 ml of medium were seeded onto plastic dishescoated with bone marrow stromal cells (plastic/BMS), plastic coated withfibronectin (plastic/FN), or into Cellfoam. Primate bone marrow stromalcells grown for 2-3 weeks to isolate the heterogeneous adherent,fibroblast-like population of cells capable of supporting HSCs inshort-term assays.

All cultures contained 1 ml of Myelocult medium (Stem Cell Technologies,Vancouver, Canada), a medium for long-term HPC culture. No exogenouscytokines were added to this medium. After 1, 1.5, 3 and 6 week ofculture as above with weekly medium changes, all cells (adherent andnon-adherent) were harvested from all culture conditions/reactors,counted, and surface antigen stained. We recovered adherent cellsbecause some primitive HPCs or HPC subclasses may exhibit adherentproperties which would prevent their being harvested by simple washingor centrifugation. Non-adherent cells were harvested from Cellfoam bysimple centrifugation for 10 minutes at 1500 rpm (approximately 250×G)in a table top centrifuge. Adherent cells were harvested with anon-trypsin isolation solution (Cell Dissociation Solution, Sigma, St.Louis, Mo.) to minimize alteration of surface staining characteristics.To recover adherent cells from Cellfoam, units were washed twice byimmersion into PBS, saturated by brief vortexing in an excess of CellDissociation Solution, incubated for 20 minutes at 37° C., andcentrifuged at 1500 rpm for 10 minutes. Non-adherent cells wererecovered from plastic/stroma and plastic/FN systems by gentle washing;adherent cells were isolated using the Cell Dissociation Solution asdescribed above. Antibodies used for surface phenotype determinationwill include anti-CD34 (Qbend10, Immunotech), anti-CD38 (OKT10, ATCC,Bethesda, Md.) and anti-CD45 (Becton Dickinson) antibodies to evaluateprogenitor cell distributions. Flow cytometry analysis of the cells wasperformed using multi-parameter FACScan flow cytometry analysis.Appropriate controls included matched isotype antibodies to establishpositive and negative quadrants, as well as appropriate single colorstains to establish compensation. For each sample, at least 10,000 listmode events were collected.

Colony-Formation Assays:

To determine whether HPCs cultured in Cellfoam for up to six weeksretain the ability to produce myeloid and erythroid colonies, weperformed traditional methylcellulose assays. Equal numbers of cellswhich have been isolated from Cellfoam, plastic/BMS or plastic/FNcultures, as described above, were added at 1×10⁴/ml to 3.0 ml ofmethylcellulose medium with cytokines (IL-3 20 ng/ml; GMCSF 30 ng/ml;erythropoietin 3 IU/ml; stem cell factor 50 ng/ml; all Stem CellTechnologies, Vancouver) plus 0.5 ml of DMEM (2% FCS, 10 IU/mlpenicillin, 10 μg/ml streptomycin, 1 mM L-glutamine). 1.5 ml of thismixture was added to a scored petri dish using a syringe and a bluntneedle to avoid bubbles. Duplicate assays were performed for eachcondition. The two duplicate petri dishes were then placed in anincubator with 5% CO₂ at 37° C. for 10-21 days. After 10-21 days, thenumber of colonies were determined by manual counting. Positive colonieswere scored on the basis of an accumulation of 20 or more cells.Erythroid colonies were scored after 14-21 days on the basis of agold-brown pigment, demonstrating hemoglobin, whereas myeloid colonieswere identified by their predominantly transparent appearance. Countswere done in duplicate.

T-Cell Lymphopoiesis:

The ability of cultured HPCs to foster T-cell lymphopoiesis was assessedin an in vitro T-cell differentiation assay in which cells isolated fromCellfoam or other cultures are seeded onto thymic stroma tissue andevaluated for the ability to produce mature T cells as assessed by CD4and CD8 single positivity and CD4CD8 double positivity antibodystaining. The T-cell differentiation assay utilizes a bed of primatethymic stromal cells plated into 24 well plates to support thedifferentiation of hematopoietic progenitor cells into thymocytes and Tcells (see U.S. Pat. No. 5,677,139, incorporated in its entirety hereinby reference). In this assay, thymic monolayer cultures are preparedfrom third trimester or neonatal rhesus thymic tissue by mincing tissueand then digesting into a single cell suspension using collagenase andDNAase. Thymic stroma cell suspensions, which can be used either freshor from cryopreserved samples, are then plated into 24 well plates.After two days, the non-adherent cells are removed and the adherent celllayer washed vigorously to remove any loose cells. After 6 days inculture, the isolated culture cells are added to the monolayer. After10-14 days, the cultures are evaluated for the presence of immaturedouble positive lymphocytes (CD3⁺CD4⁺CD8⁺), and mature single positivelymphocytes (CD3⁺CD4⁺CD8 ⁻ and CD3⁺CD4⁻CD8⁺).

In parallel for all experiments, dual controls consisting ofunfractionated bone marrow and CD34⁺ cells, neither of which had beencultured in Cellfoam, were evaluated for colony-forming potential and Tlymphopoiesis in the assays described above. The overall number ofcolonies indicates the relative number of stem cells present inCellfoam, plastic or bone marrow stroma cultures that retained theability to produce differentiated erythroid or myeloid colonies in thepresence of cytokines.

LTCIC Assays and LTCIC Transduction:

As an indicator of the ability of Cellfoam to support cells which havelong-term repopulating potential, modified LTCIC assays were performed.LTCICs are relatively quiescent cells that exhibit the characteristic ofprolonged survival in bone marrow stroma cultures, and it is during thistime that they gradually acquire the phenotype required to give rise toerythroid and myeloid colonies in vitro. An important goal of theproposed research is to determine the utility of Cellfoam in supportingthe retroviral transduction of LTCICs in vitro. These cells arerelatively quiescent, and thus have been difficult to transduceefficiently. Enhanced transduction may be facilitated by performingbiweekly transductions of the cells in Cellfoam over extended periods.Cellfoam cultures were as described above and inoculated with 2×10⁵cells, and half-volume medium exchange were performed twice a week withhigh titer retroviral supernatant (PG13LN, from ATCC, grown in an ACScartridge, titer of 1×10⁶ CFU/ml). The PG13LN vector is prepared asfollows: the retroviral producer cell line is inoculated into acartridge with up to 1800 cm² of surface and which is separated fromcirculating tissue culture medium by a semi-permeable membrane with amolecular weight threshold of 10,000 kd. Continuous circulation ofmedium through the extracapillary space by a peristaltic pump optimizesgas and nutrient exchange resulting in significant increases inretroviral vector production. Average increases in end-point dilutiontiter of retroviral vector supernatants produced using the bioreactorversus tissue culture flasks are 10-20 fold, with 100 fold increasesnoted in some instances. The infectious titer of the retroviralsupernatants produced in the continuous perfusion cartridges isdetermined via plaque forming assays on COS cells.

In addition to retroviral medium exchanges, an additional mediumexchange with LTCIC medium was performed once a week. Traditional LTCICcultures utilizing prepared bone marrow stroma acted as controls andwere cultured and transduced for the same period of time as parallelCellfoam cultures. We also attempted to culture LTCICs in plastic wellscoated with fibronectin. All culture volumes were identical. Followingtransduction in each device, methylcellulose CFU assays were performed.Total cells having undergone transduction in each device wereresuspended in 3 ml of methylcellulose medium with the addition of thecytokines IL-3 (20 ng/ml), stem cell factor (50 ng/ml), erythropoietin(3 IU/ml) and GMCSF (30 ng/ml), all part of the methylcellulose assay,and re-plated in 35-mm dishes in the presence or absence of the neomycinanalog 418 (400-800 μg/ml). After two weeks, colonies were scored usingthe criteria described above. The colony counts indicate the survival ofLTCICs after the initial six-week culture period. The relative survivalwith G418 versus without G418 indicates the survival of LTCICs that hadbeen transduced during the initial culture periods. The presence ofthese cells serves as a measure of the survival of long-termrepopulating cells, LTCICs, and their relative level of transduction inCellfoam versus plastic/BMS and plastic/FN. It is important to note thatthe initial culture of cells in Cellfoam for 6 weeks defines thetraditional threshold at which LTCICs are measured. Thus, culturing for3 or 6 weeks in Cellfoam, followed by 6 weeks in bone marrow stromaLTCIC assays extends the classic definition of LTCICs.

Example 1

We performed extended-culture survival studies examining CD34+ HPC cellnumbers at 1, 3, and 6 weeks in the absence of supplemented cytokines.Cultures were carried out in fibronectin coated Cellfoam units andcompared with bone marrow stroma and fibronectin coated plastic dishesCD34+ HPCs cultured in Cellfoam without cytokine supplementationexhibited enhanced survival and marked enrichment compared to parallelcontrol cultures. The loss of HPCs in control systems supportsdocumentation of their inability to support HPCs without exogenouscytokines. Plastic dish cultures performed similar to BMS. Conversely,at 1 week CD34+ cell counts in Cellfoam were 2.5-3 fold higher thanother systems analyzed and had increased 80-110% over input numbers. By3 and 6 weeks, as many as 6 to 10 times CD34+ cells were detected inCellfoam versus controls. This increase in cell number was reproducibleand in the absence of cytokines. In addition, we were able to count animmature population of cells (phenotype CD34+CD38−) which was enrichedin Cellfoam compared to bone marrow stroma cultures at 3 and 6 weeks;results are shown in FIG. 3 (3 weeks-1st column, 6 weeks-2nd column).

Example 2

In addition, we evaluated the multipotency of the population of cellsisolated from multi-week cytokine cultures. The assays used wereconventional methylcellulose colony-formation assays to evaluate myeloidand erythroid colony-forming cells and a published lymphopoiesis assayto evaluate T cell precursor activity. We observed that HPCs isolatedfrom Cellfoam cultures retain red blood cell (RBC) and white blood cell(WBC) colony forming ability to a greater extent than parallel controlcultures. In all cultures the CFU-GM and BFU-E were evaluated; themyeloid:erythroid ratio was approximately 2:1. At 3 weeks, Cellfoamcultures yielded up to 31 times as many colonies compared to controls,an increase of 16 fold over input capabilities (see FIG. 4). By 6 weeks,HPCs had lost essentially all of their colony-forming ability in BMS andplastic-fibronectin cultures. HPCs from Cellfoam is displayed a 1000fold greater capacity to produce colonies over control-isolated cells(see FIG. 4).

Example 3

The ability of cultured HPCs to foster T-cell lymphopoiesis was assessedin an in vitro T-cell differentiation assay. After termination ofCellfoam and control cultures at 3 and 6 weeks, an aliquot of thecombined adherent/non-adherent factions were co-cultured with primaryfetal thymic stroma. We evaluated the ability to produce mature T cellsas assessed by CD4 and CD8 single positivity and CD4CD8 doublepositivity antibody staining. When cells were harvested at 3 and 6 weeksfrom Cellfoam and control cultures and placed in the T-cell assay, onlycells recovered from Cellfoam generated T-cell progeny at both timepoints. Cells recovered from FN/plastic failed to generate T-cellprogeny. Cells from BMS cultures generated T-cell progeny at 3 weeks butnot at 6 weeks. Progeny derived from Cellfoam included CD4+CD8+thymocytes, as well as CD4+ and CD8+ cells. Progeny derived fromCellfoam cultures included CD4+CD8+thymocytes as well as CD4⁺ and CD8⁺single positive cells while most of these thymocytes express CD3, anadditional indicator of efficient T-cell development. To date, no invitro culture system has been shown to efficiently and reproduciblysupport the maintenance of an HPC population that includes T-cellprogenitors. As the assessment of multipotency is generally limited tothe generation of myeloid and erythroid colonies, the evaluation ofT-cell progeny greatly enhances our estimation of the true nature ofcells cultured long-term in Cellfoam. As the data demonstrate, Cellfoamwas able to support T-cell progenitor survival to a greater extent thancontrols. Importantly, Cellfoam provides an effective long-term culturesystem for the maintenance of multipotent HPCs ex vivo.

Example 4

We also examined the ability of Cellfoam to support the survival ofLTCICs, cells which may represent more immature hematopoieticprogenitors critical to host reconstitution. These studies utilizedLTCICs (longer surviving progenitors from cultures up to 14 weeks) thatwere subsequently plated onto traditional LTCIC plates consisting ofirradiated BMS cells. We found that HPCs isolated from fibronectincoated Cellfoam maintained LTCIC over the initial 3 week culture period(9 weeks total in culture). Cellfoam cultures yielded 17.5 times as manyLTCICs as BMS cultures. Cellfoam cultures not coated with fibronectinyielded a 4 fold increase in LTCIC activity versus BMS cultures. Thesedata suggest that Cellfoam maintains LTCIC activity to a greater extentthan control systems. This provides additional evidence that Cellfoam isadvantageous for the culture of HPCs because long-term surviving cellsare believed to be an important indicator of primitive hematopoieticprogenitor content. Six weeks cultures were followed by 6 weeks in LTCICassays and 2 weeks in colony assays, cells from plastic culturesproduced no LTCICs). Similarly, BMS cultures had lost all viable ECHCPs.However, Cellfoam cultures yielded encouraging LTCIC numbers yielding,on average, 36 times as many LTCICs as BMS cultures. Cellfoam produced18+/−8 LTCICs per 10⁴ cells compared to 0.5+/−0.7 LTCICs per 10⁴ cellsfor BMS cultures (p=0.05, n=6). Fibronectin-coated Cellfoam unitsimproved ECPHC preservation approximately 2 fold over uncoated Cellfoamunits. Uncoated units yielded 8+/−11 LTCICs per 10⁴ cells, a 16 foldincrease over BMS controls. Compared to the 3 week timepoint, the 6 weektimepoint maintained approximately half as many LTCICs in Cellfoam. Thissuggests that static cultures have a finite ability to maintain longterm culture cells or that selection of more immature, long lived cellsis ongoing. It is imperative to note that the maintenance of this numberof LTCICs at 11 and 14 weeks represents a significant breakthrough inthe culturing of HPCs. It has been reported that there is a correlationbetween the maintenance of long-lived cells and primitive HPCs whichincludes a subset of cells which may be important contributors ofself-renewal and long-term host reconstitution. As our previous datademonstrate, cells cultured over long periods in Cellfoam also retainmultipotency, a further indication that the Cellfoam system mayrepresent an enabling technology for providing the most primitive stemcells required for optimal bone marrow transplantation and repopulationof ablated hosts.

Example 5

Additionally, we have examined the transduction of colony forming HPCs,using a Neomycin resistance gene in a PG13LN retroviral vector over a 3day period. Transduction of colony-forming progenitors in Cellfoam is atleast 40-50% more efficient than BMS or plastic systems). Similarly, thetransduction efficiency of LTCICs using Cellfoam is improved by about40-50%. LTCICs were cultured in Cellfoam or BMS for 6 weeks. Retroviraltransduction was performed weekly. After 6 weeks, cells were harvestedand plated in methylcellulose with and without G418 to assesstransduction efficiency. Cells from BMS were unable to produce coloniesin the presence or absence of neomycin analog. Conversely, in Cellfoam,we obtained colonies in both G418+ and G418− assays, indicating thatLTCIC activity was preserved in Cellfoam in the presence of theretrovirus and that LTCIC transduction could be performed on these cellsin Cellfoam with 50% efficiency.

Example 6

We also examined the effect of low level cytokine supplementation onlong-term HPC survival and multipotency by culturing hematopoietic cells(including CD34+ cells and immature CD34+38− cells) on Cellfoam. Weobserved that supplementation with cytokines at levels far below thoseused in prior art results in increased hematopoietic cell numbers andcolony forming activity and maintainance and expansion of immatureprogenitors. This is in contrast to what research in the field hasshown, namely that high levels of cytokines may alter long-term HPCsurvival and multipotency. Therefore, the ability to use picogram andnanogram levels of cytokines on HPCs cultured on Cellfoam affords theopportunity, for the first time, to expand HPCs without altering theirmultipotency/function. As will be evident to those of ordinary skill inthe art, the invention enables the use of particular cytokines in thenanogram/ml and picogram/ml concentration range to achieve reproducible,practical gains in HPC number and functionality. This-unexpectedcapability has not been possible with other 2-dimensional and3-dimensional systems of the prior art. The studies described belowutilized the following concentrations of cytokines: cytokine level:nanogram (ng) level picogram (pg) level IL-3 10 ng/ml 100 pg/ml IL-6 10ng/ml 100 pg/ml FLK2 25 ng/ml 250 pg/ml SCF 25 ng/ml 250 pg/mlNote:Combination cytokines used constituent cytokines each at theconcentration shown.

In the experiments described here, an average of 45,000 CD45+ HPCs cellswere inoculated into the culture systems, cultured for one, three or sixweeks in Cellfoam or in bone marrow stroma (BMS) or plastic well controlsystems in the presence of the indicated cytokines and then evaluatedfor cell numbers and multipotency in colony formation assays. Allcultures were performed at least in quadruplicate. Particular emphasiswas placed on the yield of CD45+, CD45+34+ and CD45+34+38− cells; totalcell number was viewed as less meaningful since BMS cultures werepre-seeded with a high number of stromal cells which obfuscated totalcell number analysis. Cells were harvested, combining non-adherent andadherent fractions from single wells, and stained withfluorochrome-conjugated monoclonal antibodies to CD45 (to gate on CD45+hematopoietic cells and preclude stromal cells from analyses) and toCD34 and CD38 progenitor surface molecules.

In studies examining the effects of nanogram and picogram concentrationsof single cytokines on HPC survival in Cellfoam as compared to bonemarrow stroma (BMS), IL-3 and IL-6 showed the greatest cell expansion atthree weeks, followed by a decline at six weeks, whereas SCF and FLK2showed continued expansion from three to six weeks. All four cytokinesgenerated significantly higher cell numbers than input in Cellfoamdevices at least at one time point but only IL-3 did so in BMS (FIG. 5,top nanogram, bottom picogram concentrations). Picogram concentrationstended to show consecutive increases in CD45+ cell number from one tothree to six weeks (FIG. 5). Combinations consisting of three cytokineseach (IL-3+IL-6 and either SCF or FLK) led to increase in cell numberssimilar to single cytokines (FIG. 6, top nanogram, bottom picogramconcentrations). Similar findings in CD45+ cell counts were obtained forstudies comparing Cellfoam and plastic dish cultures.

CD45+34+ and CD45+34+38− cell numbers also tended to be higher inCellfoam than in BMS (FIG. 7, top nanogram, bottom picogramconcentrations). Of 32 possible comparisons of cell number v. singlecytokine-concentration-time datum points in Cellfoam and BMS cultures atthree and six weeks, higher numbers of CD45+34+ and CD45+34+38− cellswere observed in Cellfoam in 25 (78%). Of 16 possible comparisons ofcell number v. combination cytokine-concentration-time datum points inCellfoam and BMS at three and six weeks, higher CD45+34+ and CD45+34+38−numbers were observed in Cellfoam in all 16 (100%) (see FIG. 7 forrepresentative CD45+34+ patterns). Statistically significant values arenoted with an asterisk in FIG. 7 which compares the fold difference incell number between Cellfoam and BMS (and plastic; see below). Barsabove the 1.00 line indicate the fold higher numbers obtained inCellfoam as compared to controls; bars below the 1.00 line indicate thefold higher numbers obtained in controls as compared to Cellfoam. Scaleis shown on log base for convenience. Asterisks denote statisticallysignificant values. Patterns of fold difference for CD45+34+38− cellswere similar to those shown here for CD45+34+ cells.

Similar results to the above were obtained in comparisons of Cellfoamand plastic cultures. Of 24 possible comparisons of cell number v.cytokine-concentration-time datum points at three and six weeks, highernumbers of CD45+34+ and CD45+34+38− cells were observed in Cellfoam in21 (88%). Of 16 possible datum points for combination cytokine cultures,Cellfoam yielded more cells than plastic in 15 (94%; FIG. 7). Thus,overall, of 88 possible datum points, Cellfoam cultures yielded highernumbers in 77 (88%). In summary, these data support the conclusion thatconcentrations of cytokines in concentrations far lower than can be usedin conventional systems and which have been used routinely by previousinvestigators can be effectively used in Cellfoam to increase HPCnumber. In general, concentration of cytokines between 0.1-0.5 ng/mlpromote maintainance of HPCs, while cytokine concentrations higher thanabout 0.5 ng/ml promote differentiation of HPCs.

The function of cells cultured under these conditions was measured byevaluating in vitro colony forming capabilities utilizingmethylcellulose CFU assays. In comparing Cellfoam to plastic cultures,with the exception of IL-3 supplementation colony activity was uniformlygreater in Cellfoam than in plastic, ranging from approximately 3 to 36times greater (FIG. 8). Total CFU activity was derived by multiplyingthe colony count per 10,000 input cells by the factor for totalprogenitor number obtained in the three and six week cultures. Controlcultures added no cytokines. Thus, Cellfoam yielded both higher cellnumbers and higher colony activity than plastic cultures. It is alsointeresting to note that nanogram concentration cytokine supplementationled to decreases in total colony activity from three to six weeks (withthe exception of SCF in Cellfoam cultures) suggesting a time-dependentexposure effect of cytokine augmentation on HPC function. In picogramconcentration supplementation experiments using combination cytokines,the drop-off in CFU activity was much less dramatic, with colonyactivity remaining approximately constant from three to six weeks.Further, in certain cases, picogram levels of combination cytokines ledto higher colony activity than nanogram level supplementation. Forexample, supplementation with picogram levels of Combination 1(IL-3/IL-6/SCF) led to total colony content that was 3-6 fold higherthan with nanogram levels of Combination 1 (IL-3/IL-6/SCF) at paralleltime points.

Analysis of the fold differences in total colony activity betweenCellfoam and plastic showed that Cellfoam also generally yielded highertotal colony activity as well. With the exception of Combination 2(IL-3/IL-6/FLK2) at the six week time point, all statistically differentcolony activity values were in favor of Cellfoam in the nanogramconcentration cytokine supplementation trials (FIG. 9). Bars above the1.00 line indicate the fold higher colony numbers obtained in Cellfoamas compared to controls; bars below the 1.00 line indicate the foldhigher numbers obtained in controls as compared to Cellfoam. Scale isshown on log base. Asterisks denote statistically significant values.Picogram concentration supplementation experiments were similar tonanogram levels. Comparison of Cellfoam and BMS cultures yielded similarresults.

In summary, the experiments described above indicate that selective useof particular cytokines can lead to the expansion of colony-formingactivity as assessed by standard in vitro assays.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for in vivo maintenance, expansion and/or differentiation ofhematopoietic progenitor cells, comprising: implanting into a subject aporous, solid matrix having pre-seeded hematopoietic progenitor cellsand their progeny, wherein the porous, solid matrix is an open cellporous matrix having a percent open space of at least 75% and a unitarymicrostructure.
 2. The method of claim 1, further comprising the porous,solid matrix having pre-seeded hematopoietic progenitor cells and theirprogeny by the steps of: introducing in vitro an amount of hematopoieticprogenitor cells into the porous, solid matrix; culturing thehematopoietic progenitor cells in an environment that is free ofinoculated stromal cells, stromal cell conditioned medium, andexogenously added hematopoietic growth factors that promotehematopoietic cell maintenance, expansion and/or differentiation, otherthan serum.
 3. The method of claim 2, wherein the porous solid matrixhas pores defined by interconnecting ligaments having a diameter atmidpoint, on average, of less than 150 μm.
 4. The method of claim 3,wherein the porous solid matrix is a metal-coated reticulated open cellfoam of carbon containing material.
 5. The method of claim 4, whereinthe metal is selected from the group consisting of tantalum, titanium,platinum, niobium, hafnium, tungsten, and combinations thereof, whereinsaid metal is coated with a biological agent selected from the groupconsisting of collagens, fibronectins, laminins, integrins, angiogenicfactors, anti-inflammatory factors, glycosaminoglycans, vitrogen,antibodies and fragments thereof, and combinations thereof.
 6. Themethod of claim 5, wherein the metal is tantalum.
 7. The methodaccording to claim 1, wherein the porous, solid matrix having seededhematopoietic progenitor cells and their progeny is impregnated with agelatinous agent that occupies pores of the matrix.